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Journal of Dental Research
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Biological

{alpha}2 Integrin +807 Polymorphism in Drug-induced Gingival Overgrowth

M. Ogino1,2, J. Kido2, M. Bando2, N. Hayashi2, C. Wada2, T. Nagata2, F. Nishimura3, Y. Soga3, S. Takashiba3, T. Kubota4, M. Itagaki4, Y. Shimada4, H. Tai4, H. Yoshie4, N. Yamazaki5, Y. Shinohara1 and M. Kataoka1,*

1 Divison of Gene Expression, Institute for Genome Research, The University of Tokushima, Kuramoto 3-18-15, Tokushima 770-8503, Japan;
2 Department of Periodontology and Endodontology, Institute of Health Bioscience, Graduate School, The University of Tokushima;
3 Department of Pathophysiology/Periodontal Science, Okayama University Graduate School of Medical and Dentistry;
4 Divison of Periodontology, Department of Oral Biological Science, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan; and
5 Faculty of Pharmaceutical Science, University of Tokushima, Japan;

Correspondence: * corresponding author, kataoka{at}genome.tokushima-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
{alpha}2 integrin on fibroblasts is reported to play an important role in the induction of drug-induced gingival overgrowth, which is characterized by excessive accumulation of type I collagen in gingival connective tissue. Silent polymorphism 807 T/C within the {alpha}2 integrin gene is associated with high/low {alpha}2 integrin expression. The aim of this study was to test the hypothesis that expression of {alpha}2 integrin 807 T/C polymorphism correlates with drug-induced gingival overgrowth. A case-control study comparing 136 subjects taking calcium channel blockers (72 with vs. 64 without drug-induced gingival overgrowth) demonstrated that the frequency of the +807 C allele was significantly higher in the case group than in the controls (odds ratio, 3.61; 95% confidence interval, 2.14 – 6.10; P < 0.05). The present findings suggest that the {alpha}2 +807 C allele is one of the genetic risk factors for drug-induced gingival overgrowth.

Key Words: drug-induced gingival overgrowth • {alpha}2 integrin • SNP • collagen phagocytosis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Drug-induced gingival overgrowth is an adverse effect associated with 3 types of drugs: phenytoin, an anti-epileptic drug; cyclosporin A, an immunosuppressant drug; and calcium channel blockers that have been widely prescribed for treatment of various cardiovascular diseases, mostly hypertension, such as dihydropyridines (i.e., nifedipine), diltiazem, and verapamil (Nishikawa et al., 1996). Gingival overgrowth is characterized by an accumulation of extracellular matrix within the gingival connective tissue, particularly the collagenous components (Yamasaki et al., 1987). The metabolism of collagen, the most abundant protein in mammals, is precisely balanced by collagen synthesis and degradation to maintain a steady state (Perez-Tamayo, 1978). Fibrosis is caused by loss of this collagen fiber homeostasis (Korn et al., 1992). Collagen fibrils are degraded via an extracellular pathway involving secretion of collagenase (Murphy and Reynolds, 1985), and via an intracellular pathway involving phagocytosis in fibroblasts (Everts et al., 1985). Collagen phagocytosis is thought to be an important pathway for physiological degradation of collagen in gingival connective tissue (Sodek and Overall, 1988). Inhibition of collagen phagocytosis by fibroblasts is one of the mechanisms leading to gingival overgrowth (McCulloch and Knowles, 1993; Kataoka et al., 2000, 2001).

Integrins are the principle mediators of molecular dialogue between a cell and its extracellular matrix environment (Gumbiner, 1996). The unique combinations of integrin subunits determine which extracellular matrix molecules will be recognized by a cell. The function of the {alpha}2β1 integrin on the platelet has been well-studied, and the availability of the {alpha}2β1 integrin on the platelet surface plays an essential role in platelet adhesion to collagen in vessel walls (Saelman et al., 1994). Furthermore, an ’807 C to T single nucleotide’ exchange polymorphism in the gene encoding the {alpha}2 subunit of the {alpha}2β1 integrin is known to be associated with an increase in the density of the {alpha}2β1 integrin on the platelet surface, as well as with increased platelet adhesion to collagen (Jacquelin et al., 2001; Kunicki, 2002). The {alpha}2β1 integrin serves as a specific receptor for type I collagen in fibroblasts (Dickeson et al., 1999), and the initial binding step of collagen phagocytosis relies on adhesive interaction between fibroblasts and collagen. The {alpha}2 integrin plays a critical role in the phagocytic regulation of collagen internalization (Chou et al., 1996; Lee et al., 1996; Lee and McCulloch, 1997), and drug-induced gingival overgrowth is caused by inhibition of collagen phagocytosis by a reduction of {alpha}2 integrin expression in fibroblasts, as demonstrated in a rat experimental model (Kataoka et al., 2003). Genetic polymorphism can cause unexpected outcomes, such as therapeutic failure, toxicity, and adverse effects in subjects undergoing medical treatment (Kim et al., 2004). These findings have led to the hypothesis that subjects with the 807 C genotype of the {alpha}2 integrin express less {alpha}2β1 integrin on the gingival fibroblast surface, thus reducing the potential for fibroblast binding to type I collagen and collagen phagocytosis in response to drugs, and in turn increasing the risk of drug-induced gingival overgrowth. In this study, for early identification of individuals at risk for drug-induced gingival overgrowth, we investigated whether {alpha}2 integrin polymorphism is associated with calcium-channel-blocker-induced gingival overgrowth.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
One hundred thirty-six subjects who had been receiving calcium channel blockers for hypertension for at least 1 yr took part in this study. The study was approved by the Institutional Review Board of Tokushima University School of Medicine, and written informed consent was obtained from all patients, in accordance with the Helsinki Declaration, before their inclusion in the study. The subjects were divided into two groups according to established periodontal criteria (McGaw et al., 1987). McGaw et al.(1987) reported that the patients with a gingival overgrowth score grade 1 (blunting of gingival margin) or less (no overgrowth, feather-edged gingival margin) were regarded as non-responders (without gingival overgrowth). Grade 2 is moderate gingival overgrowth (< 1/3 of crown length), and grade 3 is marked gingival overgrowth (> 1/3 of crown length); patients with grades 2 and 3 were regarded as responders (with gingival overgrowth). In gingival overgrowth, we could often find gingival inflammation, which acts as an exacerbating factor for gingival overgrowth. Thus, it is relatively difficult to distinguish between scores 2 and 3 clinically. The purpose of this study was to investigate whether {alpha}2 integrin polymorphism is associated with the induction of drug-induced gingival overgrowth. We divided the subjects into two groups, those without gingival overgrowth (grade 1 or less) and those with gingival overgrowth (grade 2 or more): 64 subjects (29 men, 35 women; mean age, 68 yrs; range, 42–88 yrs) without (grade 1 or less) and 72 subjects (37 men, 35 women; mean age, 62 yrs; range, 8–83 yrs) with gingival overgrowth (grade 2 or more).

DNA Analysis
Genomic DNA was obtained from peripheral venous blood samples after standard venipuncture. All blood samples were collected in sterile tubes containing potassium EDTA. DNA was extracted by means of a DNA extraction kit (Takara Shuzo, Kyoto, Japan) according to the instruction manual. A DNA fragment containing the site of {alpha}2 integrin 807 T/C polymorphism was amplified by PCR with a specific primer set (5'-gtgtttaacttgaacacatataaaaccaa-3' and 5'-gaaggcagctgggaaagttaaatc-3') based on the sequence data (GenBank accession number AF035968). After the 310-bp DNA fragment had been purified by electrophoresis on 1.5% agarose gel, and the subsequent use of a DNA purification kit (QIAGEN GmbH, Hilden, Germany), nucleotide sequencing was performed by cycle sequencing coupled with primers designed for the amplification of DNA with Gene Rapid (Amersham, Arlington Heights, IL, USA).

Statistical Analysis
We used the {chi}2 test to compare differences between the case and control allele or genotype frequencies. Association between the allele and gingival overgrowth was assessed by calculation of the odds ratio and 95% confidence intervals (CI).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Nifedipine-induced Gingival Overgrowth in the Anterior Facial Area
The calcium channel blocker induced gingival overgrowth (Fig.Go). The enlarged fibrous gingival tissue covered the facial surfaces of the teeth, particularly in the interdental areas.


Figure 1
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  		Figure. Gingival overgrowth in anterior facial area of a 60-year-old woman after 2 years of nifedipine treatment for hypertension.

 
Allele and Genotype Frequencies of the +807 Polymorphism within {alpha}2 Integrin in Subjects with or without Gingival Overgrowth
We analyzed a total of 136 DNA samples, from 72 drug-induced gingival overgrowth patients and 64 controls, for +807 polymorphism within the {alpha}2 integrin gene. The frequencies of the allele and genotypes of +807 polymorphism within the {alpha}2 integrin gene are shown in the TableGo. The CC and TC genotypes were present in 59.7% and 36.1% of the subjects with gingival overgrowth, respectively, whereas, in the control group, the corresponding frequencies were 29.7% and 39.1% (P = 2.17 x 10–5). Furthermore, a significant difference was observed between the case group (77.8%) and the control group (49.2%) in the frequency of the +807 C polymorphic allele (P = 9.20 x 10–7; odds ratio = 3.61, 95% CI = 2.14 – 6.10). These results indicated that the +807 polymorphism within the {alpha}2 integrin gene is a strong genetic factor involved in drug-induced gingival overgrowth.


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  		Table. Allele and Genotype Frequencies of the +807 Polymorphism within {alpha}2 Integrin in Subjects with or without Gingival Overgrowth (n = 136)
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The incidence of drug-induced gingival overgrowth has been reported to vary: 10–50% for phenytoin, 8–70% for cyclosporin A, and 0.5–83% for calcium channel blockers (Nishikawa et al., 1996). These variations may be caused by many factors, such as blood drug levels and/or duration of drug intake and the severity of growth, sex predilection, and effect of local gingival inflammation. The genetic capacity of the host to deal metabolically with administered drugs, the responsiveness of gingival tissue to the drugs, and the pre-existing condition of the gingiva, including gingival inflammation—which is an exacerbating factor for drug-induced gingival overgrowth—may differ among individuals. Overall, the manifestation of a drug’s side-effect is known to be mostly dependent on gene polymorphism.

The {alpha}2 integrin is known to play an important role in the induction of drug-induced gingival overgrowth (Chou et al., 1996; Lee et al., 1996; Lee and McCulloch, 1997). Inhibition of collagen phagocytosis by reduction of {alpha}2 integrin expression in gingival fibroblasts induces excessive collagen accumulation in gingival connective tissue, which in turn leads to gingival overgrowth (Kataoka et al., 2003). Interestingly, {alpha}2 integrin expression on platelets is highly variable, even among normal individuals, and there is, on average, a five-fold range of platelet {alpha}2β1 integrin density, associated with the inheritance of at least 3 {alpha}2 gene alleles, that influences the degree of platelet adhesion to collagens (Kunicki et al., 1993, 1997; Kritzik et al., 1998; Roest et al., 2000; Jacquelin et al., 2001; Kunicki, 2002). The {alpha}2 gene 807 T is associated with a high density of platelet {alpha}2β1, whereas allele 807 T/C and 807 C are associated with lower densities (Kunicki et al., 1993, 1997; Kritzik et al., 1998; Roest et al., 2000; Kunicki, 2002). Thus, it is conceivable that {alpha}2 gene alleles might directly affect changes in the density of {alpha}2β1 integrin on gingival fibroblasts and collagen phagocytosis in gingival connective tissue. We therefore speculated that the {alpha}2 integrin 807 T/C polymorphism may be related to the induction of drug-induced gingival overgrowth, and that {alpha}2 integrin 807 C may predispose individuals to gingival overgrowth through low expression of the {alpha}2β1 integrin on gingival fibroblasts. This would have more severe inhibitory effect on drug-induced collagen phagocytosis than that of 807 T, leading to excessive accumulation of collagen fibers in gingival connective tissues. With regard to the low density of the {alpha}2 integrin on fibroblasts resulting from {alpha}2 integrin 807 C, its expression level would be easily reduced to below the threshold of collagen phagocytosis for induction of gingival overgrowth by drugs. Recently, Di Paola et al.(2005) showed that the frequency of 807 T in a Japanese population is 0.41. The 807 T frequency of 0.508 in controls in this experiment is evidently higher than that of the other report. The reason for this frequency is unknown. However, if a control frequency of 0.41 were used, there would be 75 807 C and 53 807 T chromosomes, and the difference would still be statistically significant (P = 0.001). Likewise, the control genotypes would be 22 CC, 31 CT, and 11 TT, and the difference would still be significant (P = 0.003). The {alpha}2 integrin 807 T/C polymorphism does not affect the amino acid sequence of the {alpha}2 integrin protein. Although we did not examine {alpha}2 integrin expression on gingival fibroblasts in this study, linkage with other functionally relevant polymorphisms of the {alpha}2 integrin gene seems plausible. This is the first study to demonstrate an association between {alpha}2 integrin polymorphism and drug-induced gingival overgrowth.

In summary, analysis of our data provides evidence that the {alpha}2 integrin gene plays an etiologically important role in drug-induced gingival overgrowth, and that the frequency of the {alpha}2 integrin 807 C allele is significantly increased in subjects with drug-induced gingival overgrowth. Drug-induced gingival overgrowth may be reduced by the elimination (with dental plaque control and periodontal maintenance therapy) of exacerbating factors such as dental plaque and calculus, but not prevented in the susceptible patient. The most effective treatment of drug-induced gingival overgrowth is withdrawal or substitution of medication. The present findings will be useful for the early identification of individuals at risk for drug-induced gingival overgrowth, and may also provide a basis for the selection of medications to prevent it.


   ACKNOWLEDGMENTS
 
This study was supported by grants in aid (16592068 and 16209062) for scientific research from the Japan Society for the Promotion of Science.

Received for publication April 28, 2005. Revision received August 9, 2005. Accepted for publication August 30, 2005.


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 DISCUSSION
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Journal of Dental Research, Vol. 84, No. 12, 1183-1186 (2005)
DOI: 10.1177/154405910508401217
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